Project Summary: Podocyte foot processes and their interposed slit diaphragms form the final barrier to urinary protein loss which explains the typical association of podocyte injury with proteinuria. The degree of podocyte loss correlated with disease severity in several kidney disorders including diabetic nephropathy and IgA nephropathy Our recently published data has identified dendrin as a novel slit diaphragm component that relocates to the podocyte nucleus to enhance apoptosis under the influence of transforming growth factor beta (TGF-beta). We have found that dendrin relocates to the nucleus in a mouse model of focal segmental glomeruloclerosis where TGF-beta levels are increased. In this application we have proposed two specific aims to study the regulation of podocyte survival by dendrin. In Specific Aim 1 we will elucidate the molecular mechanism regulating the relocation of dendrin from the slit diaphragm to the podocyte nucleus. We will utilize confocal microscopy to test whether loss of alpha-actinin-4 increases the nuclear expression of dendrin. We will test whether TGF-beta induces the phosphroylation of dendrin by an in vivo phorphorylation assay. Then we will induce point mutations of putative binding sites to test whether 14-3-3 binds to dendrin in a phosphorylation-dependent manner to promote nuclear import. In Specific Aim 2 we will determine how dendrin enhances TGF-beta induced podocyte apoptosis. The effect of overexpressing Yes associated Protein and Nedd4-2 on TGF-beta induced apoptosis will be measured by an Annexin binding assay and FACS analysis. Subsequently we will cross dendrin null mice with CD2AP null and TGF-beta transgenic to determine whether the loss of dendrin ameliorates the severity of glomerulosclerosis in these two mouse models where TGF-beta expression is increased. Relevance: The proposed studies seek to determine the mechanism whereby our protein of interest, dendrin, enhances injury and programmed death of kidney visceral epithelial cells or podocytes. Injury to these cells is an important mechanism in the progression of proteinuric kidney diseases. PUBLIC HEALTH RELEVANCE: This work seeks to elucidate the details of an important mechanism leading to injury and death of critically important cells (podocytes) lining the urinary space. These findings could have an impact on the development of novel therapeutic agents for kidney diseases such as diabetic nephropathy, focal segmental glomeruloclerosis and IgA nephropathy, all disorders associated with a reduction in podocyte number.